Antivirus antibiotic from paecilomyces todicus and method of producing same



1967 B. SOKOLOFF ETAL 3,303,094

ANTIVIRUS ANTIBIOTIC FROM PAECILOMYCES TODICUS I AND METHOD OF PRODUCINGSAME Filed Aug. 27, 1962 3 Sheets-Sheet l Paecllomyces todicus. U.V. aeed 5 Inn A Scale 0-]. X Sens. 3B1 Period .2 2.5 mg./1 m1.' 1 g 2 PM 20XPBS FIG.

PAECILOMYIIIJS 'IOUICUS wavenumber in Kaysers I I I I I I I I I IWavelength in Microns Speed 1 Scale 2 in/p Gain 3.76 Period 2 $11!:Sched 2X ordinate Scale T SB DB BONS SOKOLOFF YOSWHARU TOUR anew-4g 1957B. SOKOLOFF ETAL. 3,303,094

ANTIVIRUS ANTIBIOTlC FROM PAECILOMYCES TODICUS AND METHOD OF PRODUCINGSAME Flled Aug. 27, 1962 5 Sheets-Sheet z no.5 GMT 1 A A U U U Solvent:H 0. Developin Solvent: (1)

- Acetone; (2 chloroform; f3) Ethyl alcohol;

(4) Methyl alcohol; (5) Butyl alcohol. Spot: 1% methylene blue alcoholsolution, rinsed with alcohol. Detecting spot is not stained butbackground in atainecl blue.

F\C,,4 CHART 2 Fs S CHART 3 NO. 28

Method: olvent: H 0, Developing Solvent: Butyl alcohol part glacialacetic acid 1 part, H 0 2 parts. pot: Methylene blue.

Method: (1) Solvent: H 0; (2) Developing solvent: Butyl alcohol parts;Glacial acetic acid 1 part; H 0 2 parts; (3) S ot: 3 0.2% Ninhydrlnsolution. BOPJS SOKD D YOSHINRN) TOUR 1967 B. SOKOLOFF ETAL 3,

ANTIVIRUS ANTlBIOTlC FROM PAECILOMYCES TODICUS AND METHOD OF PRODUCINGSAME Flled Aug. 27, l962 3 Sheets-Sheet 3 6 CHART MOUSE SABCOHA 180After 8 days, tumor weight in grams CHART '3 EHRLICH MOUSE CARCINOMACONTROL Grams u TREATED After 8 days, tumor waight in grams 3 BORISSOKOLOFF T Yosun-umu TODB United States Patent 3,303,094 ANTIVIRUSANTIBIOTIC FROM PAECILOMYCES TODICUS AND METHOD OF PRODUCING SAME BorisSokololf and Yoshiharu Toda, both of Florida Southern College, Lakeland,Fla. 33803 Filed Aug. 27, 1962, Ser. No. 219,614 3 Claims. (Cl. 167-65)This invention relates to a new and useful active antiviral antibioticsubstance, isolated from a new species of the genus of Paecilomyces, andto the method of its extraction and purification.

Since the discovery of penicillin, many new antibiotics have beenisolated and a number of them are being used in the treatment ofinfectious diseases. As yet no active antiviral antibiotic has beendiscovered. In our search for such an antibiotic, we investigatednumerous fungi by the method of endobiosis in chick embryos. Afterhaving tested many fungi, we came across a mold which has shown a strongactivity against influenza virus PR8 and influenza virus MP1, ine-mbryonated eggs. After having isolated a crude antibiotic compound, itwas tested in vivo in mice against the same viruses and also against thetransplanted tumors mouse sarcoma 180 and Ehrlich mouse carcinoma. Theantibiotic showed a strong inhibiting activity against transplantedtumors. This unique property of the antibiotic, to be active againstboth viruses and tumors indicates its potential usefulness, particularlyin view of its low toxicity.

It is accordingly a fundamental object of this invention to provideantiviral, antitumor compound in useful form and a method of preparingit.

The antibiotic substance is obtained from a mold, originating in theFlorida Everglades swamp, which we identified as belonging to the genusof Paecilomyces. Upon comparing the biological, morphological andchemical properties of other species of this genus, as described byBrown and Smith in their monograph on this genus, published in theTransactions of the British Mycological Society, 40:17-89, 1957, it wasestablished that the mold, discovered by us, is a new one and differsfrom any other members of this genus. Accordingly, it was calledPaecilomyces todicus, nov. species, and has been deposited with and isavailable from the Culture Collection Section, Fermentation Division,Southern Regional Research Laboratory, of the United States Departmentof A-griculture, and incorporated into the permanent culture collectionof microorganisms as NRRL 2975.

The outstanding characteristics of the mold are:

Conidial fructification: Branched from curved hyphae consisting ofseparate sterigmatic cells. Sterigmata regularly distributed along thefertile hyphae.

Sterigmata: to 15 microns by 2 microns with short tubes usually straightfrom the axis of the cells, diverged at the apices, bearing short chainsof conidia.

Conidia: Oval, 3 to 5 by 1 to 2 microns, brown, a little rough-walled,swelling in germination to 5-6 microns and producing many tubes.

Heat resistance of spores: 60 C. for 15-20 minutes.

The mold does not grow on nutrient agar, brain-heartinfusion agar,Barnes media, and corn-steep media.

' The growth of it is increased remarkably by adding cornsteep water tosynthetic media containing dextrose.

It prefers dextrose to saccharose.

It does not take lactose, adonit, sorbit, inosit, inulin, and dextrin asthe sugars.

It grows both on acid and neutral media, but prefers acid media.

It produces yellow pigment of an extracellular nature, on various media.The colony is white to brownishwhite. It is never colored greenish onany media.

Utilization of nitrogen: The surface growth of the mold is very thick oncasein hydrolysate and corn-maize. The growth is very thin onbacto-asparagin, l-proline and altryptophane. It almost does not grow onnutrient broth, polypeptone, neopeptone, soytone, yeast extract,brainheart-infusion.

It grows best on the media containing: Glucose 40.0 gm.; K HPO 1.0 gm.;NaNO 3.0 -gm.; MgSO 0.5 gm.; KCl, 0.5 gm; FeSO 0.001 gm.; corn-maize,10.0 ml.; water, 1000 ml.; pH 6.0-6.4.

There are considerable fluctuations in the pH during the fermentationperiod. With the initial pH of 6.0, it goes down to pH 3.5 on the fifthday and up to pH 6.0 on the 11th day, 7.0 on the 14th day. When theinitial pH 6.8, it goes down to 4.2 on the 7th day and up to 6.8 on the19th day.

Search for references related to the antibiotic produced by the genusPaecilomyces revealed several [Vora, V. A. Metabolic ProductsPaecilomyces victoriae. J. Sci. Ind. Research (India) 13B, 842-4, 1954.;Yonehara, H., Takeuchi, S., Umezawa, H. and Suzuki, Y. Variotin, a NewAnt'ifungal Antibiotic, produced by Paecilomyces varioli var.antibiotic-us. J. Antibiotics (Japan) Ser. A, 12, 109- 10, 1959; Kenner,G. W. and Sheppard, R. C., a-aminoisobutyric acid, B-hydroxyleucine, and'y-methylproline from Hydrolysis of a Natural Product. Nature 181, 48,1958.; Suzuki, 8., Suganuma, A., Kishida, T Misunoya, A., Ueda, F.,Suzuki, Y., Kiji, 1., Kyo, T., Takeuchi, M., Nakazawa S., Kishi N. andTsumura, 8., Activity of Variotin, A New Antifun-gal Antibiotic. I.Antibiotics (Japan) Ser. A 12, 244-8, 1959.; Saksena, S. B., A NewSpecies of Paecilomyces from Soil, Journ. Indian Bot. Soc., 32(4):186-189, 1953.1

M. Pisano, A. Fleischman, and M. Littman: Antibiotic Production byPaecilomyces persicinus. Antimicrobial Agents Annual, 1241-7, 1960.

A. Fleischman and M. Pisano: The production of synnemartin B byPaecilomyces persicinus in a chemically defined medium. AntimicrobialAgents Annual, 1:48- 53, 1960.

One of the antibiotics produced by the species of the Paecilomyces genuscorrespond in their biological activity to that of Paecilomyces todicus.As our tests, described below, have shown the antibiotic produced byPaecilomyces todicus has no activity against any microorganisms, eitherbacteria or fungi, while all the antibiotics produced by other speciesof the genus of Paecilomyces so far described are active against eitherbacteria of fungi.

The mold we have isolated is aerobic. It grows well both on the surfaceand immersed in a nutrient medium. Both the mycelium and the mediumcontain the active factor after a period of fermentation. The modifiedCzapek-Dox medium to which corn-maize was added served us for productionof the antibiotic. On this liquid medium, the mold starts to grow after24 hours, with the submerged mycelium reaching the surface. The optimaltemperature is around 27 to 30 C. At 37 it grows.

somewhat faster in the beginning but soon the growth slows and isstopped. After growing for 10 days at the temperature of 27-30" C., thesurface of the culture (around 200 cm?) is filled with mycelium. Thesurface Patented Feb. 7, 1967 a 4 is first white, then a few spaces ofyellow colored mate- TABLE TESTS ON THE ANTIBIOTIC rial appear on themedium surface. The surface by that Reactions: R lt time is irregularlyconvex and concave. Ninhydrin The antibiotic is extracted from a l12 dayo Molischs T821565]; n face culture, but it can be isolated also fromthe culture 5 Vaninin and Hcl media less or more aged. A diluted acetone(75%) is the most convenient method for the initial extraction of theactive factor, although methanol or ethanol alcohols can be us d l f thipurpose The antibiotic was tested on various bacterial and molds withnegative results. Table II summarizes the results.

TABLE II.EFFECT OF THE ANTIBIOIIC ON BACTERIA AND FUNGI Quanibo(oxidized nitroprusside) Keto-l-uronic acid Microorganisms Doses Pstudo-St. aureus E. coli Proteus monas C vulgan's aeruginosa albtcans Themethod of extraction is as follows: The results presented in Table IIindicate that the anti- The mycelium is macerated and extractedrepeatedly 25 biotic produced by Paecilomyces todicus exerts neither anwith several times its volume of acetone (75-25% antimicrobial orantifungal activity. Similarly, the medium is extracted. All thefractions ob- The antiviral activity of the antibiotic was tested firsttained both from the mycelium extractions and medium in embryonated eggson influenza virus PR8 and on inextractions, are combined. The activefactor is precipifluenza Avirus MFl. tated with Ba(OH) The waterextraction of Ba(OH) The strain of influenza virus was suspended innutrient precipitate is lyophilized, and the material is purified bybroth at pH 7.0 and diluted by the 10-fold dilution repeated extractionswith acetone and ethanol. Instead method. The antibiotic was dissolvedin distilled water of lyophilization, the active factor can be separatedfrom at pH 7.0 at the rate of 0.1, 0.2 and 0.5 mg./0.1 ml. for theBarium precipitate by extraction with dilute acetone, testing. The virussuspension of each dilution was inocu- 2540%, precipitated with 95%acetone, and washed lated into the chorio-allantoic cavity of aten-day-old several times with ethanol. Aluminum oxide might be chickembryo, and thereafter the antibiotic was injected used instead ofBa(OH) The antibiotic so obtained is into the same site of theembryonated eggs and incubated in the form of a powder of a yellowishcolor, soluble in for 24 hours. After refrigerating over night, thechoriowater, diluted acetone, and alcohols. allantoic fluids were pooledfrom each embryonated egg In FIGURE I, we have shown the UV. absorptionand titrated by the modified Salk pattern test. Table III curve, andsummarizes the results of testing various batches of the In FIGURE II wehave shown wavelength plotted antibiotic. against absorption, for thepowder TABLE III.THE ACTIVITY OF THE ANTIBIOIIC AGAINST INFLUENZA AVIRUSES PR8 AND MFI IN EMBRYONATED EGGS Influenza Virus PR8D osesInfluenza Virus MFl-Doses Batches 0.1 mg. 0.2 mg. 0.5 mg. 0.1 mg. 0.2mg. 0.5 mg.

ID Control 3 #28 ID50 Treated 6.5 6.32 I 6.0 6. 2 6.1 5.8 ID50 Control 8#29 ID50 Treated 6.2 5.33 5. 34 6.0 I 5.75 5.4 ID50 Control s ID50Treated 5. 6 5. 3 5, 0 5. 9 5. 5 5. 1

Paper chromatography data on the antibiotic are sum- Although there aresome variations in the biological marized in FIGURES III, IV, V;activity of the antibiotic against influenza viruses in eggs, FIGURES VIand VII summarize some mouse test usually present in any biologicaltesting, there is a definite, data. well-pronounced activity against thetwostrains of In- With Water as solvent, and acetone, chloroform, ethylfluenza A virus. While the control shows ID50 above alcohol, methanoland butyl alcohol as developers, there 8 (8 the ID50 for the treatedeggs fluctuates between was no actual development as is shown in FIGUREIII. 6.5 and 5.0 depending on the dose administered.

Using water as. solvent and as the developing agent: The antibioticcures mice infected with influenca A butyl alcohol 4 parts, glacialacetic acid 1 part and H 0 virus, PR8 and MP1. Influenza A virus PR8 andMP1 2 parts, and staining with 1% methylene blue, there was adapted tomouse lung infection through three passages certain development as shownon FIGURE IV. was used. On the third day after the last passage, eachWhen water was used as solvent and the same developinfected lung withinfluenza virus was removed aseptically ing agents were used but 0.2%ninhydrine was applied for and macerated finely with sand in a smallamount of spotting, a different development was recorded as shown Tyrodesolution with penicillin. After centrifuging for on FIGURE V. tenminutes at 2000 r.p.m., the supernatant was separated Identity testsgiving clues to the chemical structure from sediment and used forinoculation. Each mouse of the antibiotic are as follows: was inoculatedintranasally with 4 drops of virus suspension under ether. Sixteen hoursafter virus infection, the antibiotic in various daily doses wasinjected intraperitoneally for four or five days. On the 14th day aftervirus inoculation, each surviving mouse was killed and the lungs removedindividually. They were homogenated with sand and a small amount ofphosphate buffer saline at pH 7.0. The homogenate was centrifuged forminutes at 2000 r.p.m. and the supernatant was used for titration. Themodified Salk pattern test was used with 0.5% chick blood cellsuspension. The results of treatment of the infected mice are summarizedin Table IV.

TABLE IV.-ACTIVITY OF THE ANTIBIOTIC AGAINST INFLUENZA A VIRUS, PR8 ANDMFl IN MICE Infectivlty Rate, Percent Virus Dose Total Mortality Rate,Percent Influenza A virus PR8:

Control. I 100 Treated 6.0 rug/mouse ln 4 50 days. Control 100 Treated8.0 rug/mouse in 4 days. Control 100 Treated 10.0 mg./mouse in 4 10days. Influenza A virus MFl:

Control- 100 Treated 6.0 rug/mouse in 4 55 days. Control 100 Treated 8.0rug/mouse in 4 days. Control 95 Treated 10.0 mg./mouse in 5 12. 5

days. Control 95 Treated 12.0 mg./mouse in 5 7. 5

days.

The mice cured from influenza with the antibiotic showed to be immuneagainst the infection with the same strain of virus.

The antibiotic exerts a strong tumor inhibiting activity. The usualstandard method for testing tumor inhibition was used. The mice, 24, 48,or 72 hours after the tumor was transplanted, were given the antibiotic,injected int-raperitoneally for seven days, with the control groupwithout any treatment. On the ninth day after tumor transplantation, alltumors, in the treated and control groups, were removed, and theirweight recorded. The average tumor weight of the treated group wascompared with the control tumor.

The following examples illustrate antitumor activity of the antibiotic.

Thirty mice, vmales, weighing average 20.5 gm. were divided into twogroups of fifteen animals each. One group served as control and theother group was given the antibiotic. To all mice, sarcoma 180 wastransplanted and 48 hours after transplantation, the treatment wasstarted. It continued -for 7 days, with the total dose of antibioticgiven of 14 mg. or 700.0 rng./kg. wt./mouse. On the ninth day, all thetumors were removed and weighed. The average weight in the control groupwas 3.44 gm., in the treated group 0.65 gm. as shown on FIGURE VI.

To another thirty mice, Ehrlich mouse carcinoma was transplanted andwhen the transplant was 48 hours old,

2. Process for the production of antiviral substance which comprises,under aerobic conditions, cultivating a culture of Paecz'lo mycestodicus nov. sp. NRRL 2975 at temperature of 15 to 40 C. in an aqueousnutrient medium containing sources of assimilable nitrogen and carbonand then extracting the mycelium and medium with acetone, precipitatingactive factors from the acetone extract with Ba(OH) extracting theprecipitate with a solvent comprising water, lyophilizing the aqueousextract and extracting the lyophilized product with a solvent comprisingacetone, to leave a yellow powder residue of antiviral substance.

3. Process for the production of antiviral substance which comprises,under aerobic conditions, growing a culture of Paecilomyces todicus nov.sp. NRRL 2975 at a temperature of 15 to 40 C. in an aqueous nutrientmedium containing sources of assim-ilable nitrogen and carbon, withessential minerals added, and then extracting the mycelium and mediumwith acetone, precipitating active factors from the acetone extract withBa(OH) and extracting the precipitate with water and acetone, to obtaina yellow powder residue of antiviral substance.

No references cited.

SAM ROSEN, Primary Examiner.

JULIAN S. LEVITI, Examiner.

1. AN ANTIVIRAL MATERIAL PRODUCED BY THE PROCESS OF FERMENTATION OF ACULTURE OF PAECILOMYCES TODICUS, NOV. SP. NRRL 2975, IN A NUTRIENTMEDIUM UNDER AEROBIC CONDITIONS, SAID MATERIAL BEING A WATER-SOLUBLE,POWDER OF A LIGHT YELLOW COLOR, SAID MATERIAL BEING EXTRACTED FROM THEMYCELIUM AND MEDIUM BY MEANS OF ACETONE AND RECOVERED FROM THE ACETONEEXTRACT BY BARIUM HYDROXIDE PRECIPITATION.